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- EMDB-47832: Structure of nanobody AT206 in complex with the losartan-bound an... -

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Basic information

Entry
Database: EMDB / ID: EMD-47832
TitleStructure of nanobody AT206 in complex with the losartan-bound angiotensin II type I receptor (AT1R)
Map dataLocal map of AT206-AT1RBril-losartan complex
Sample
  • Complex: Nanobody AT206 in complex with the losartan-bound angiotensin II type I receptor (AT1R)
    • Protein or peptide: Nanobody AT206,Type-1 angiotensin II receptor,Soluble cytochrome b562
    • Protein or peptide: BAG2 Anti-BRIL Fab Heavy Chain
    • Protein or peptide: BAG2 Anti-BRIL Fab Light Chain
  • Ligand: [2-butyl-5-chloranyl-3-[[4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: CHOLESTEROL
KeywordsGPCR / AT1R / nanobody / MEMBRANE PROTEIN
Function / homology
Function and homology information


angiotensin type I receptor activity / positive regulation of phospholipase A2 activity / angiotensin type II receptor activity / phospholipase C-activating angiotensin-activated signaling pathway / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin / bradykinin receptor binding / renin-angiotensin regulation of aldosterone production / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / : ...angiotensin type I receptor activity / positive regulation of phospholipase A2 activity / angiotensin type II receptor activity / phospholipase C-activating angiotensin-activated signaling pathway / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin / bradykinin receptor binding / renin-angiotensin regulation of aldosterone production / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / : / low-density lipoprotein particle remodeling / positive regulation of macrophage derived foam cell differentiation / positive regulation of protein metabolic process / regulation of systemic arterial blood pressure by renin-angiotensin / Rho protein signal transduction / regulation of vasoconstriction / Peptide ligand-binding receptors / blood vessel diameter maintenance / kidney development / angiotensin-activated signaling pathway / cell chemotaxis / regulation of cell growth / calcium-mediated signaling / electron transport chain / positive regulation of inflammatory response / positive regulation of reactive oxygen species metabolic process / Cargo recognition for clathrin-mediated endocytosis / regulation of cell population proliferation / Clathrin-mediated endocytosis / positive regulation of cytosolic calcium ion concentration / regulation of inflammatory response / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / periplasmic space / electron transfer activity / G protein-coupled receptor signaling pathway / inflammatory response / iron ion binding / protein heterodimerization activity / symbiont entry into host cell / heme binding / membrane / plasma membrane
Similarity search - Function
Angiotensin II receptor type 1 / Angiotensin II receptor family / : / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM ...Angiotensin II receptor type 1 / Angiotensin II receptor family / : / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
Soluble cytochrome b562 / Type-1 angiotensin II receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSkiba MA / Liu J / Kruse AC
Funding support United States, 4 items
OrganizationGrant numberCountry
Helen Hay Whitney Foundation United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)K99HD110612 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)R21HD101596 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA260415 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Epitope-directed selection of GPCR nanobody ligands with evolvable function.
Authors: Meredith A Skiba / Clare Canavan / Genevieve R Nemeth / Jinghan Liu / Ali Kanso / Andrew C Kruse /
Abstract: Antibodies have the potential to target G protein-coupled receptors (GPCRs) with high receptor, cellular, and tissue selectivity; however, few antibody ligands for GPCRs exist. Here, we describe a ...Antibodies have the potential to target G protein-coupled receptors (GPCRs) with high receptor, cellular, and tissue selectivity; however, few antibody ligands for GPCRs exist. Here, we describe a generalizable selection method to enrich for GPCR ligands from a synthetic camelid antibody fragment (nanobody) library. Our strategy yielded multiple nanobody ligands for the angiotensin II type I receptor (AT1R), a prototypical GPCR and important drug target. We found that nanobodies readily act as allosteric modulators, encoding selectivity for both the receptor and chemical features of GPCR ligands. We then used structure-guided design to convert two nanobodies from allosteric ligands to competitive AT1R inhibitors through simple mutations. This work demonstrates that nanobodies can encode multiple pharmacological behaviors and have great potential as evolvable scaffolds for the development of next-generation GPCR therapeutics.
History
DepositionNov 11, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47832.png

Downloads & links

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Map

FileDownload / File: emd_47832.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal map of AT206-AT1RBril-losartan complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 352 pix.
= 292.16 Å		0.83 Å/pix.
x 352 pix.
= 292.16 Å		0.83 Å/pix.
x 352 pix.
= 292.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.085
Minimum - Maximum-0.7091434 - 1.079641
Average (Standard dev.)0.00003793199 (±0.013170009)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 292.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Global J68 Global map of AT206-AT1RBril-losartan complex, half map

Fileemd_47832_additional_1.map
AnnotationGlobal J68 Global map of AT206-AT1RBril-losartan complex, half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Global J68 Global map of AT206-AT1RBril-losartan complex, half map

Fileemd_47832_additional_2.map
AnnotationGlobal J68 Global map of AT206-AT1RBril-losartan complex, half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Global J68 Global map of AT206-AT1RBril-losartan complex

Fileemd_47832_additional_3.map
AnnotationGlobal J68 Global map of AT206-AT1RBril-losartan complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Local map of AT206-AT1RBril-losartan complex, half map

Fileemd_47832_half_map_1.map
AnnotationLocal map of AT206-AT1RBril-losartan complex, half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Local map of AT206-AT1RBril-losartan complex, half map

Fileemd_47832_half_map_2.map
AnnotationLocal map of AT206-AT1RBril-losartan complex, half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Nanobody AT206 in complex with the losartan-bound angiotensin II ...

EntireName: Nanobody AT206 in complex with the losartan-bound angiotensin II type I receptor (AT1R)
Components
  • Complex: Nanobody AT206 in complex with the losartan-bound angiotensin II type I receptor (AT1R)
    • Protein or peptide: Nanobody AT206,Type-1 angiotensin II receptor,Soluble cytochrome b562
    • Protein or peptide: BAG2 Anti-BRIL Fab Heavy Chain
    • Protein or peptide: BAG2 Anti-BRIL Fab Light Chain
  • Ligand: [2-butyl-5-chloranyl-3-[[4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: CHOLESTEROL

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Supramolecule #1: Nanobody AT206 in complex with the losartan-bound angiotensin II ...

SupramoleculeName: Nanobody AT206 in complex with the losartan-bound angiotensin II type I receptor (AT1R)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Nanobody AT206,Type-1 angiotensin II receptor,Soluble cytochrome b562

MacromoleculeName: Nanobody AT206,Type-1 angiotensin II receptor,Soluble cytochrome b562
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.542836 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLQESGGG LVQAGGSLRL SCAASGSISY YRMGWYRQAP GKEREFVAGI GVGTTTNYAD SVKGRFTISR DNAKNTVYLQ MNSLKPEDT AVYYCAAYNY FPRSIVYYYV YWGQGTQVTV SSGGSGEDQV DPRLIDGKIL NSSTEDGIKR IQDDCPKAGR H NYIFVMIP ...String:
QVQLQESGGG LVQAGGSLRL SCAASGSISY YRMGWYRQAP GKEREFVAGI GVGTTTNYAD SVKGRFTISR DNAKNTVYLQ MNSLKPEDT AVYYCAAYNY FPRSIVYYYV YWGQGTQVTV SSGGSGEDQV DPRLIDGKIL NSSTEDGIKR IQDDCPKAGR H NYIFVMIP TLYSIIFVVG IFGNSLVVIV IYFYMKLKTV ASVFLLNLAL ADLCFLLTLP LWAVYTAMEY RWPFGNYLCK IA SASVSFN LYASVFLLTC LSIDRYLAIV HPMKSRLRRT MLVAKVTCII IWLLAGLASL PAIIHRNVFF IENTNITVCA FHY ESQNST LPIGLGLTKN ILGFLFPFLI ILTSYTLIWK ALKKAYDLED NWETLNDNLK VIEKADNAAQ VKDALTKMRA AALD AQKAT PPKLEDKSPD SPEMKDFRHG FDILVGQIDD ALKLANEGKV KEAQAAAEQL KTTRNAYIQK YLERARSTLD KLNDD IFKI IMAIVLFFFF SWIPHQIFTF LDVLIQLGII RDCRIADIVD TAMPITICIA YFNNCLNPLF YGFLGKKFKR YFLQLL KYG GSSLEVLFQG PTETSQVAPA

UniProtKB: Type-1 angiotensin II receptor, Soluble cytochrome b562, Type-1 angiotensin II receptor

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Macromolecule #2: BAG2 Anti-BRIL Fab Heavy Chain

MacromoleculeName: BAG2 Anti-BRIL Fab Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 24.539314 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCD

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Macromolecule #3: BAG2 Anti-BRIL Fab Light Chain

MacromoleculeName: BAG2 Anti-BRIL Fab Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.541164 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: [2-butyl-5-chloranyl-3-[[4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]ph...

MacromoleculeName: [2-butyl-5-chloranyl-3-[[4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol
type: ligand / ID: 4 / Number of copies: 1 / Formula: LSN
Molecular weightTheoretical: 422.911 Da
Chemical component information

ChemComp-LSN:
[2-butyl-5-chloranyl-3-[[4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol / medication*YM

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 6 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 4322 / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1748565
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 379250
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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